Use of monoclonal antibodies in cancer immunotherapy: types and mechanisms of action.

Immunotherapy is one of the most innovative treatments in the current field of oncology and consists of stimulating the immune system to eliminate tumoral cells. Monoclonal antibodies (mAbs) are glycoproteins secreted by B-cells capable of recognizing and neutralizing foreign organisms or antigens. Structurally, they are composed of two heavy and two light chains. The generation of therapeutic mAbs is one of the most developed and fastest-growing areas of the biotechnological and pharmaceutical industries and is an important adjunct to cancer therapy. Several antibodies have been approved for human administration and can be mouse-derived, chimeric, humanized, or fully human. mAbs main mechanism of action includes the lysis of the tumoral cells through inducing apoptosis, phagocytosis, complement activation, or signaling inhibition.

La inmunoterapia es un tratamiento innovador para la oncología actual, que consiste en la estimulación del sistema inmunitario para la eliminación de las células tumorales. Los anticuerpos monoclonales (mAbs) son glicoproteínas secretadas por los linfocitos B, capaces de reconocer y neutralizar organismos extraños o antígenos. Estructuralmente se componen de dos cadenas pesadas y dos cadenas ligeras. La generación de mAbs terapéuticos es una de las áreas de mayor crecimiento en la industria biotecnológica y farmacéutica y representa un complemento importante en la terapia del cáncer. Existen diversos mAbs que han sido aprobados para su administración en humanos, y pueden ser derivados de ratón, quiméricos, humanizados o completamente humanos. Los mecanismos de acción consisten principalmente en la lisis de las células tumorales a través de la inducción de la apoptosis, fagocitosis, activación del complemento o inhibición de la señalización celular.

Use of monoclonal antibodies in cancer immunotherapy: types and mechanisms of action / Uso de anticuerpos monoclonales en la inmunoterapia del cáncer: tipos y mecanismos de acción

Abstract Immunotherapy is one of the most innovative treatments in the current field of oncology and consists of stimulating the immune system to eliminate tumoral cells. Monoclonal antibodies (mAbs) are glycoproteins secreted by B-cells capable of recognizing and neutralizing foreign organisms or antigens. Structurally, they are composed of two heavy and two light chains. The generation of therapeutic mAbs is one of the most developed and fastest-growing areas of the biotechnological and pharmaceutical industries and is an important adjunct to cancer therapy. Several antibodies have been approved for human administration and can be mouse-derived, chimeric, humanized, or fully human. mAbs main mechanism of action includes the lysis of the tumoral cells through inducing apoptosis, phagocytosis, complement activation, or signaling inhibition.

Resumen La inmunoterapia es un tratamiento innovador para la oncología actual, que consiste en la estimulación del sistema inmunitario para la eliminación de las células tumorales. Los anticuerpos monoclonales (mAbs) son glicoproteínas secretadas por los linfocitos B, capaces de reconocer y neutralizar organismos extraños o antígenos. Estructuralmente se componen de dos cadenas pesadas y dos cadenas ligeras. La generación de mAbs terapéuticos es una de las áreas de mayor crecimiento en la industria biotecnológica y farmacéutica y representa un complemento importante en la terapia del cáncer. Existen diversos mAbs que han sido aprobados para su administración en humanos, y pueden ser derivados de ratón, quiméricos, humanizados o completamente humanos. Los mecanismos de acción consisten principalmente en la lisis de las células tumorales a través de la inducción de la apoptosis, fagocitosis, activación del complemento o inhibición de la señalización celular.

Nanophotonic Sialidase Immunoassay for Bacterial Vaginosis Diagnosis.

Bacterial vaginosis (BV) affects reproductive-age women and can lead to pelvic inflammatory disease, postpartum endometritis, and preterm labor/delivery and predisposes the infection of sexually transmitted diseases. Typically, BV diagnosis involves the analysis of vaginal swab samples via microscopy operated by highly skilled personnel. Hence, novel approaches for BV diagnosis are an existing need. In response, the first immunosensing platform targeting sialidase, a BV biomarker, is reported. The nanophotonic operational principle of this biosensing platform allows for a cheaper, faster, and simpler analysis when compared with an indirect enzyme-linked immunosorbent assay (ELISA). The clinical evaluation of such a nanotechnology is highlighted, where 162 vaginal swab samples were analyzed with high sensitivity and specificity (96.29%, respectively). The resulting nanoimmunosensing platform offers a resourceful approach to perform a timely BV diagnosis.

Production and characterization of a monoclonal antibody against the sialidase of Gardnerella vaginalis using a synthetic peptide in a MAP8 format.

Bacterial vaginosis is one of the most frequent vaginal infections. Its main etiological agent is Gardnerella vaginalis, which produces several virulence factors involved in vaginal infection and colonization, in particular, sialidase (SLD), a potential clinical biomarker that participates in immune response modulation and mucus degradation. The main objective of this work was the production and evaluation of a monoclonal antibody against G. vaginalis sialidase and its validation in immunoassays. For immunization of mice, a synthetic multiantigenic peptide was used, and hybridomas were generated. After fusion, hybridomas were evaluated for antibody production and cloned by limited dilution. One clone producing IgG1 was selected and characterized by indirect ELISA, dot blot, and Western blot, and we also tested clinical isolates and HeLa cells infected with G. vaginalis. The results showed that the anti-SLD antibody recognized a single protein of ~90 kDa that correlated with the estimated molecular weight of SLD. In addition, anti-SLD antibody recognized SLD from complete bacteria and from culture supernatants of infected Hela cells. In conclusion, our results showed that the anti-SLD antibody recognized SLD from different sources and could be considered a new tool for the diagnosis of bacterial vaginosis. KEY POINTS: • Anti-sialidase mAb was generated using a synthetic peptide • The mAb recognizes synthetic peptide and intact protein from multiple sources • The antibody was characterized by several immunological methods.

Production and characterization of monoclonal antibodies against the DNA binding domain of the RE1-silencing transcription factor.

The use of monoclonal antibodies for the detection of cellular biomarkers during carcinogenesis provides new strategies for cancer diagnosis or prognosis in patients. Loss of the Restrictive Element 1-Silencing Transcription (REST) factor has been observed in previous molecular and immunological approaches in aggressive breast cancer, small cell lung cancer, liver carcinoma, and colo-rectal cancer; however, for clinic diagnosis, monoclonal antibodies for REST recognition are unavailable. The goal of this work was to design, produce and characterize monoclonal antibodies against the REST DNA binding damain (DBD) that would be suitable for immunoassays. We searched for conserved domains, and immunogenic and antigenic sites in the REST structure via in silico analysis. For mice immunization, we used a recombinant REST DBD purified by affinity chromatography, and then Hybridomas were generated by mouse spleen fusion with myeloma cells. Finally, for monoclonal antibody characterization, we performed enzyme-linked immunosorbent (ELISA), western blot, dot blot, immunocytochemistry (ICC) and immunoprecipitation assays. Results showed that the DBD is conserved in REST isoforms and contains immunogenic and antigenic sites. We generated three clones producing monoclonal antibodies against REST DBD, one of them specifically recognized native REST and was suitable for ICC in samples from patients.

Theranostic-PDT with the antibody anti isoform 4 SOD mitocondrial labeled with PpIX in the lung cancer cell line A-549.

BACKGROUND: In this work, a drug product composed of an IgM antibody derived from a hybridoma subclone 4C1F6D5G7B8 was prepared and further labeled with PpIX to be used in cell lines A-549 and MRC-5. The aim of this work was to evaluate the potential theranostic activity of the obtained product together with photodynamic therapy (PDT). METHODS: The IgM antibody labeled with PpIX was used in different concentrations to perform theranostics with PDT in cell lines A-549 and MRC-5 in order to identify the specificity of IgM antibody in lung cancer cells by means of a LED-irradiation system set at 630 nm. The location of the conjugate was further determined by confocal microscopy. RESULTS: The theranostic with conjugate Ab-PpIX in the A-549 cell lines showed fluorescence by confocal microscopy, whereas the MRC-5 cell line showed no reactivity. The PDT with the conjugate in the cell line A-549 decreased its viability 70% compared to the control. On the contrary, with the MRC-5 cell line no viability diference was shown. The confocal microscopy applied to the cell line A-549 showed that the Ab-PpIX was majorly located at the cytoplasm. CONCLUSION: Ab-PpIX showed therapeutical potential in lung cancer cells A-549 and had no activity in non-cancerous lung cells (MCR-5).

Successful capture of Toxocara canis larva antigens from human serum samples.

BACKGROUND: Toxocara canis is a nematode that parasitizes dogs, while humans are paratenic hosts. When humans are infected the migrating larvae damage the liver, lungs and even the nervous system. Larva migrans diagnosis is based on immunological techniques; however, the commercial immunodiagnostic kits detect anti-T. canis antibodies which may cross-react with other parasites, mainly nematodes with extra-intestinal migration. Moreover, antibodies do not necessarily reflect an active infection; so detection and quantification of circulating antigens may provide appropriate and timely information for treatment, which prevents irreversible damage. Here we report the standardization of a monoclonal antibody based antigen capture ELISA to diagnose human toxocariasis without cross-reaction. METHODS: We developed anti-T. canis polyclonal antibodies in rabbits and a monoclonal antibody in mouse which did not cross-react with 15 antigens from several parasites. The sandwich ELISA standardization was performed using sera from mice experimentally infected. We tested the method using 29 positive and 58 negative human sera previously typified with a commercial kit, which detects antibodies. RESULTS: Only 5.0 µg/mL and 10 µg/mL polyclonal antibodies and monoclonal antibody, respectively, were needed in the sandwich ELISA standardization, detecting since 440 pg/mL larva antigens. Nine out of 29 antibody-positive sera were also positive for antigens and no false positive were found. Taking the antibody kit as the reference standard, the sensibility and specificity of the antigen test were 31% and 100%, respectively. CONCLUSIONS: With these tools we established a detection threshold as low as 440 pg/mL antigen. Monoclonal antibody is specific, and did not cross-react with antigens from other parasites. Detection of circulating antigens helps provide appropriate and timely treatment and prevents irreversible damage.

Entamoeba invadens, encystation process and enolase.

The reptilian parasite Entamoeba invadens is accepted as a model for the study of the Entamoeba encystation process. Here we describe the production and characterization of a mAb (B4F2), generated against a component of the E. invadens cyst wall. This mAb specifically recognizes a 48-kDa protein present in cytoplasmic vesicles of cells encysting for 24 h. In mature cysts (96 h), the antigen was detected on the cyst surface. By two-dimensional electrophoresis and mass spectrometry analysis, the B4F2 specific antigen was identified as enolase. Levels of enolase mRNA were increased in encysting cells and the B4F2 mAb was found to inhibit cyst formation. Therefore, these results strongly suggest a new role for enolase in E. invadens encystation, and the B4F2 mAb will be useful tool to study its role in the differentiation process.